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DOI: 10.1148/radiol.2381041668
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(Radiology 2006;238:200-210.)
© RSNA, 2006

Molecular Imaging

MR Evaluation of the Glomerular Homing of Magnetically Labeled Mesenchymal Stem Cells in a Rat Model of Nephropathy1

Olivier Hauger, MD, PhD, Emma E. Frost, PhD, Ruud van Heeswijk, BS, Colette Deminière, MD, R. Xue, PhD, Yahsou Delmas, MD, Christian Combe, MD, PhD, Chrit T. W. Moonen, PhD, Nicolas Grenier, MD and Jeff W. M. Bulte, PhD

1 From the Department of Radiology and Radiological Sciences (O.H., E.E.F., R.v.H., R.X., J.W.M.B.) and Institute for Cell Engineering (O.H., E.E.F., J.W.M.B.), Johns Hopkins University School of Medicine, Baltimore, Md; Department of Molecular and Functional Imaging, ERT CNRS/Université Victor Segalen-Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux Cedex, France (O.H., C.T.W.M., N.G.); and Anatomic Pathology Laboratory (C.D.) and Department of Nephrology (Y.D., C.C.), Hôpital Pellegrin, CHU Bordeaux, Bordeaux, France. Received October 1, 2004; revision requested November 25; revision received January 28, 2005; final version accepted February 25. O.H. supported by NIH RO1 NS 045062, Société Française de Radiologie, Institut National de la Santé et de la Recherche Médicale, and Fondation de France. Address correspondence to O.H. (e-mail: olivier.hauger{at}chu-bordeaux.fr).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Purpose: To assess renal glomerular homing of intravenously injected superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs) at in vivo and ex vivo magnetic resonance (MR) imaging in an experimental rat model of mesangiolysis.

Materials and Methods: Animal procedures were performed in accordance with protocols approved by Institutional Animal Care and Use Committee. Fourteen rats were divided into two groups: one pathologic (n = 10), with persistent mesangiolysis following simultaneous injection of OX-7 monoclonal antibody and puromycin aminonucleoside in which 107 SPIO- and DiI-labeled MSCs were injected, and one control (n = 4). In vivo and ex vivo MR imaging examinations were performed with 4.7- and 9.4-T spectrometers, respectively, and T2*-weighted sequences. In vivo signal intensity variations were measured in the liver and kidney before and 6 days after MSC injection. Intrarenal signal intensity variations were correlated with histopathologic data by means of colocalization of DiI fluorescence, {alpha}-actin, and Prussian blue stain–positive cells. Histologic differences between the glomerular homing of MSCs in different kidney portions were correlated to the areas of MR signal intensity decrease with nonparametric statistical tests.

Results: On in vivo images, signal intensity measurements of pathologic kidneys following MSC injection did not show any signal intensity decrease (P = .7), whereas a 34% ± 14 (mean ± standard deviation) signal intensity decrease was observed in the liver (P < .01), where a substantial number of labeled cells were trapped. On ex vivo images, pathologic kidneys showed focal cortical (glomerular) areas of signal intensity loss, which was absent in controls. The areas of low signal intensity correlated well with {alpha}-actin and Prussian blue stain– and DiI-positive areas (P < .01), which indicates that MSCs specifically home to injured tissue. No MSCs were detected in the kidneys of control animals.

Conclusion: Intravenously injected MSCs specifically home to focal areas of glomerular damage and can be detected at ex vivo MR imaging.

© RSNA, 2006


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Renal insufficiency is a major public health problem. Many renal diseases involving tubular and glomerular cells are irreversible and progressive and lead to chronic renal failure. Mesangial cells, a smooth muscle component of the glomerular vasculature originating from mesenchymal stem cells (MSCs), play a key role in the development of scarring in many glomerular diseases (1). However, specific therapies are lacking for most of these nephropathies, and very little is known about the potential of stem cells for their treatment. As for the latter, pluripotent and multipotent stem cells have shown great therapeutic promise because of their ability to target diseased tissue and their potential to differentiate into a wide variety of cell types (2–6). Findings of few studies have shown the ability of bone marrow-derived cells to differentiate into glomerular mesangial cells (7–9) and to deliver a modified phenotype to normal glomeruli (10) after lethal whole-body irradiation. Thus, the prospect of using bone marrow–derived stem cells, in particular MSCs, for glomerular repair may be feasible, and further studies are clearly warranted. A key component for the amelioration of nephropathy with cell-based therapy is the ability of MSCs to home to the site of tissue injury and engraft within the glomerulus. The use of noninvasive imaging techniques to assess the success of the transplant may greatly facilitate the evaluation of this therapeutic strategy.

In vivo MR monitoring of MSCs after grafting and the determination of their precise location are key components for developing MSC therapy, whether these cells are used as replacement cells or as vectors for gene therapy. By using superparamagnetic iron oxide (SPIO) particles for magnetic labeling of progenitor and stem cells, MR imaging has shown its usefulness as a noninvasive method to monitor experimental cell-based therapies. Most of the MR tracking studies have used a local administration of cells (ie, grafting) in the brain (11–16), spinal cord (17), skeletal muscle (18), or myocardium (19–21). As for systemic administration of stem cells, MSCs have been shown to localize in the liver and kidney following intraarterial injection (22). However, many strategies of stem cell therapy are based on intravenous administration of cells, as this pathway is least invasive. Few MR imaging studies have addressed the homing potential of stem cells in disease models following systemic (intravenously injected) administration (23–26). The purpose of our study was to assess renal glomerular homing of intravenously injected SPIO-labeled MSCs at in vivo and ex vivo MR imaging in an experimental rat model of mesangiolysis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Nephropathy Model
Various types of nephropathies can lead to renal failure, and some of them can be reproduced in animals with an injection of nephrotoxic substances (ie, puromycin aminonucleoside) (27) or antibodies against kidney components (ie, antithymocyte antibody) (28), which induces morphologic changes similar to those seen in immunoglobulin A nephropathy and lupus nephritis. When the antithymocyte (anti–Thy 1.1) antibody is given to rats, it binds to the glomerular mesangial cell and induces a complement-mediated cell injury. This glomerular injury is lytic at an early stage, followed by a proliferative response at a later stage. The glomerular lesions are therefore reversible in a short period of time. To obtain a persistent glomerular disturbance with a prolonged phenotypic alteration of mesangial cells, we chose a model developed by Shinosaki et al (29) that consists of a simultaneous injection of anti–Thy 1.1 antibody and puromycin aminonucleoside, which leads to injury of both mesangial and glomerular epithelial cells with progressive and irreversible renal disease. The anti–Thy 1.1 and puromycin aminonucleoside models have been used successfully in previous MR imaging studies (30,31).

Cell Culture and Labeling
Bone marrow–derived MSCs from Lewis rats were kindly provided by Dr D. J. Prockop of the Center for Gene Therapy, Tulane University Health Science Center, New Orleans, La (32) and were cultured in a standard medium (D-MEM; Sigma, St Louis, Mo) that was supplemented with 10% fetal bovine serum (Sigma), 4 mmol/L L-glutamine (Invitrogen, Carlsbad, Calif), and 500 µg/mL gentamicin (Invitrogen). Cells were cultured in 150-cm2 flasks (Falcon, Franklin Lakes, NJ) and were split at 70% confluence. Prior to injection, they were labeled with both SPIO and the fluorescent marker 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, Eugene, Ore). DiI is a lipophilic dye that labels cells by means of lateral diffusion through the plasma membrane, with an absorbance wavelength of 480 nm and emission at 565 nm. Labeled cells were identified with both fluorescence microscopy and Prussian blue staining (O.H., E.E.F., J.W.M.B).

For SPIO labeling, 48 hours prior to injection, the MSCs were grown in complete medium containing 2.2 µL/mL of SPIO (Feridex; Berlex, Wayne, NJ) and 0.25 µL/mL Superfect (Qiagen, Valencia, Calif). Superfect is a transfection agent based on a low-generation heat-activated dendrimer. Six hours before injection, the SPIO-containing medium was removed from the cells and replaced with a complete medium. Then, 1 hour prior to injection, cells were labeled with DiI at a concentration of 5 µL/ml. Immediately prior to injection, the labeled MSCs were rinsed twice in phosphate-buffered saline, were trypsinized, and were washed again. After the cells were counted with a hemocytometer, the cell pellet was resuspended in sterile 0.9% sodium chloride solution in deionized water, with a final cell concentration of 2 x 107 cells per milliliter. Cells were injected in the tail vein immediately following collection at a concentration of 8–10 x 106 cells in 0.5 mL of saline.

Animal Experiments
Animal procedures were performed by two of the authors (O.H., E.E.F.) in accordance with the protocols approved by our Institutional Animal Care and Use Committee. The animals had free access to a standard diet and water throughout the entire protocol. For all experiments, 4–6-week-old animals were anesthetized with 1%–1.5% isoflurane (Abbott Laboratories, North Chicago, Ill). The nephropathy was induced with a simultaneous intraperitoneal injection of 60 mg/kg of puromycin aminonucleoside and intravenous (tail vein) injection of 1 mg/kg of monoclonal anti–Thy 1.1 (OX-7 clone) antibody.

This study involved 14 Lewis male rats (150–250 g) divided into four groups: two pathologic groups (five animals in group 1 and five in group 2, with MSCs injected 4 and 8 days, respectively, after the induction of nephropathy) and two control groups (two animals in each group). In the first control group, nephropathy was induced but no cells were injected; in the second control group, cells were injected in normal animals (nephropathy was not induced). In all groups, the animals were sacrificed 6 days after MSC injection.

In Vivo MR Imaging
The in vivo MR imaging study consisted of two sessions in which a spectrometer operating at 4.7 T (47-50 Biospec; Brüker Instruments, Karlsruhe, Germany) was used (O.H., R.B.v.H.). For all sessions, the animals were anesthetized with 1.0%–1.5% isoflurane. Sessions were performed for all pathologic rats and included a baseline study before intravenous injection of labeled MSCs and a second study 6 days after. Animals were placed in a supine position in a 70-mm-inner-diameter birdcage volume coil (Brüker Instruments), with the positioning allowing the analysis of both the kidneys and the liver. Fast low-angle shot gradient-echo sequences (repetition time msec/echo time msec, 300/12; flip angle, 30°) were used to enhance the T2* effects of the SPIO particles. The following parameters were used: six signals acquired per image; matrix size, 256 x 256; field of view, 55 mm; section thickness, 1 mm; and total acquisition time, 7.40 minutes. The kidneys and the lower portion of the right hepatic lobe were imaged in the transverse plane.

After the second in vivo imaging session, the animals were sacrificed with a lethal intraperitoneal injection of 50 mg of sodium pentobarbital (Nembutol; Abbott Laboratories). The sternum and ribs on the left side of the chest were removed to expose the heart, and 1 mL phosphate-buffered saline (Dulbecco's formulation) with 10 U/mL heparin (Sigma) and 0.5% sodium nitrite solution (Sigma-Aldrich, St Louis, Mo) was injected directly into the left ventricle and allowed to circulate while the rest of the abdomen was opened. Immediately prior to perfusion, a small incision was made in the lateral wall of the left ventricle halfway between the atrioventricular groove and the apex of the heart. A 28-gauge needle attached to a homemade perfusion set-up was inserted into the ventricle and clamped in place. The right atrium was snipped to allow fluid to escape. Then 60–75 mL phosphate-buffered saline was perfused into the heart until the fluid from the right atrium ran clear, after which the animal was perfused with 60–75 mL 4% paraformaldehyde in phosphate-buffered saline. The organs of interest (liver and both kidneys) were removed and postfixed with immersion in 4% paraformaldehyde for at least 16 hours.

Ex Vivo MR Imaging
Ex vivo imaging studies were performed (R.X.) for all pathologic and control rats with a 400-MHz (9.4-T) imaging spectrometer (Omega; GE Medical Systems, Milwaukee, Wis). In each case, the left kidney was arbitrarily chosen for MR imaging. Before imaging, the organs were embedded in a lubricant (Fomblin; Ausimont, Thorofare, NJ) to obtain a dark background with no proton signal. A custom-made solenoid radiofrequency coil was used as both receiver and transmitter. A T2*-weighted gradient-echo sequence was used with a repetition time of 110 msec, and four echoes were obtained with echo times of 7.5, 15.7, 23.9, and 32.1 msec (the second echo was used to provide the best contrast and was further used for display of the images). An adiabatic radiofrequency pulse of 30° was used to excite the full volume. The field of view was 22 x 15 x 13 mm, with an imaging matrix of 256 x 120 x 96. The imaging resolution was 86 x 125 x 135 µm, which was zero padded to 43 x 63 x 68 µm. This three-dimensional sequence required an experimental time of 7 hours per complete three-dimensional data set, with 20 signals acquired.

Immunohistochemistry
Tissues were prepared for cryosectioning by washing them in phosphate-buffered saline for 24 hours, which was followed by cryoprotection in 30% sucrose in phosphate-buffered saline until the tissue sank to the bottom of the tube (24–72 hours). The tissue was then embedded in a special low-temperature embedding medium (Tissue-Tek OCT compound; Miles, Elkhart, NJ) for cryosectioning at 5 µm section thickness. The kidneys were sectioned in the sagittal axis for comparison with the in vivo and ex vivo MR images. After the recognition of superior and inferior poles, the poles were subdivided into the following four sections of equal size: superior, superior midportion, inferior midportion, and inferior. For each section, 20 glomeruli were analyzed for the assessment of hematoxylin-eosin-saffron staining, anti–{alpha}-actin immunostaining, Prussian blue staining for SPIO particles, and DiI fluorescence. For Prussian blue staining (O.H., E.E.F.), the sections were incubated in 1% potassium ferrocyanide (Perls reagent) solution (Sigma) containing 6% hydrochloric acid for 30 minutes in the dark (33). Slides were washed three times in phosphate-buffered saline, were transferred to a neutral fast red (Fluka) counterstain for 5 minutes, were washed in deionized water, and were mounted in a mounting medium (Permount; Fisher Scientific, Ferlane, NJ). For anti–{alpha}-actin immunostaining (O.H., E.E.F.), 5-µm sections were deparaffinized with xylene for 30 minutes and rehydrated by using graded ethanol concentrations. Antigen retrieval was performed by using steam (a heat-induced epitope retrieval method). Immunohistochemical staining was performed with the avidin-biotin-peroxidase technique and 3,3'diaminobenzidine as chromogen by using the automated BioTek system (Ventana BioTek Solutions; Tucson, Ariz). Muscle-specific anti–{alpha}-actin (Sigma) was used at 1:8000 dilution.

Assessment of hematoxylin-eosin-saffron staining was performed according to a four-grade scale: grade 0, normal; grade 1, limited segmental proliferation (<50% of glomeruli); grade 2, lobulated glomeruli with substantial proliferation (50%–75% of glomeruli); and grade 3, lobulated glomeruli with major proliferation (>75% of glomeruli). Assessment of {alpha}-actin staining was performed according to a four-grade scale: grade 0, normal; grade 1, 0%–25% of positive cells; grade 2, 26%–50% of positive cells; and grade 3, more than 50% of positive cells. Assessments of both hematoxylin-eosin-saffron and {alpha}-actin staining were performed by a pathologist (C.D.) with more than 30 years of experience in renal pathology.

Analysis of Prussian blue staining and DiI fluorescence consisted of counting positive glomeruli among those that were analyzed (O.H., C.D.). Glomeruli were considered positive when DiI-positive cells were detected at 600 nm (red channel) but not at 400 nm (green channel), which ensured the specific presence of the DiI-fluorescent cell tracker without autofluorescence. Glomeruli were considered positive for Prussian blue staining when at least one blue cell was detected.

Data Collection and Statistical Analysis
MR imaging.—For in vivo imaging, both qualitative visual analysis and signal intensity measurements were performed on the kidneys and the liver. One author (O.H.) placed three regions of interest (.025 cm2) on the three renal compartments (inner medulla, outer medulla, and cortex) and a fourth one in the lower portion of the right lobe of the liver. The results were first expressed as the mean plus or minus standard deviation of the signal intensity (SI) normalized to the noise (N) (SInN): (SInN) = ([SI – N]/N). The signal intensity values of the second imaging session were then normalized to the muscle (SInM) to cancel the signal intensity fluctuations related to variations in technical parameters between both imaging sequences. The following formula was used: SInM = SInNx (SIM1/SIM2), where SIM1 and SIM2 are signal intensity in the muscle in the first and second imaging sessions, respectively. A paired student t test was used to compare each group before and 6 days after the injection of MSCs. Differences with P value of less than .05 were considered significant. When the change in signal intensity was significant, the percentage of signal intensity decrease before (SIB) and after (SIA) injection of labeled cells was calculated according to the following formula: [(SIB – SIA)/SIA]x 100.

For ex vivo imaging, the analysis consisted of qualitative visual analysis only (O.H., N.G.), since preinjection data were obviously not available. After recognition of the superior and inferior poles, the kidneys were subdivided into four portions as previously described for the immunohistochemistry (superior, superior midportion, inferior midportion, and inferior). The areas of signal intensity decrease were first located according to this segmentation. Areas of hypointense signal intensity were then located according to the kidney compartments (cortex, outer medulla, or inner medulla) (Fig 1).



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  		Figure 1: Ex vivo sagittal three-dimensional T2*-weighted (110/15.7, flip angle of 30°) 9.4-T MR image of a control kidney. The kidney is divided into four portions of equal size: superior (1), superior midportion (2), inferior midportion (3), and inferior (4).

 
Immunohistochemistry.—In each of the four previously defined areas of the kidney, semiquantitative scores for areas of hematoxylin-eosin-saffron and {alpha}-actin staining and for Prussian blue stain– and DiI-positive glomeruli were analyzed according to the presence or absence of a signal intensity decrease on 9.4-T MR images (O.H., C.C.). Mann-Whitney U tests were considered positive at P < .05. Correlations between the immunohistochemical parameters were evaluated with a Spearman rank correlation test. All analyses were performed (O.H., C.C.) with software (Statistica 6.0; Statsoft, Tulsa, Okla). In this experimental study, we had no previous assumption about the fate of the MSCs in the diseased kidneys, and therefore no prestudy power estimations were made.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
MR Imaging
SPIO-labeled MSCs were injected in 12 rats (10 pathologic and two controls). Before injection, the cells were verified to be positive for Prussian blue staining and DiI fluorescence (Fig 2). For in vivo imaging at 4.7 T, qualitative analysis and signal intensity measurements of all pathologic kidneys did not show any signal intensity decrease in any compartment, including the cortex (P = .7). On the contrary, a significant (P < .001) signal intensity decrease was noted in the liver of all rats (normal or pathologic) in which labeled MSCs were injected (Fig 3). This signal intensity decrease appeared homogeneous and ranged from 32.1% ± 8 (average ± standard deviation) in controls to 34.3% ± 14 in pathologic rats. No signal intensity decrease was noted either in the kidneys or the liver of the control animals in which no cells were injected (Table 1).



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  		Figure 2a: In vitro assessment of MSCs colabeled with SPIO and DiI giving a red emission at 600 nm. Micrographs from (a) Prussian blue staining (original magnification, x40) and (b) fluorescent microscopy (original magnification, x40) show strong (>80% positive cells) labeling for both cell tracking markers.

 


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  		Figure 2b: In vitro assessment of MSCs colabeled with SPIO and DiI giving a red emission at 600 nm. Micrographs from (a) Prussian blue staining (original magnification, x40) and (b) fluorescent microscopy (original magnification, x40) show strong (>80% positive cells) labeling for both cell tracking markers.

 


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  		Figure 3a: In vivo transverse T2*-weighted (300/12, flip angle of 30°) 4.7-T MR images of the liver (L) and right kidney (K) (a) before and (b) 6 days after intravenous injection of 107 labeled MSCs in a pathologic rat. Note the absence of any signal intensity decrease in the right kidney and the dramatic signal intensity decrease in the liver (arrow). (c) Micrograph shows that this signal intensity decrease is related to the presence of Prussian blue–positive cells (arrows) corresponding to MSCs trapped in the sinusoids. (Original magnification, x25.)

 


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  		Figure 3b: In vivo transverse T2*-weighted (300/12, flip angle of 30°) 4.7-T MR images of the liver (L) and right kidney (K) (a) before and (b) 6 days after intravenous injection of 107 labeled MSCs in a pathologic rat. Note the absence of any signal intensity decrease in the right kidney and the dramatic signal intensity decrease in the liver (arrow). (c) Micrograph shows that this signal intensity decrease is related to the presence of Prussian blue–positive cells (arrows) corresponding to MSCs trapped in the sinusoids. (Original magnification, x25.)

 


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  		Figure 3c: In vivo transverse T2*-weighted (300/12, flip angle of 30°) 4.7-T MR images of the liver (L) and right kidney (K) (a) before and (b) 6 days after intravenous injection of 107 labeled MSCs in a pathologic rat. Note the absence of any signal intensity decrease in the right kidney and the dramatic signal intensity decrease in the liver (arrow). (c) Micrograph shows that this signal intensity decrease is related to the presence of Prussian blue–positive cells (arrows) corresponding to MSCs trapped in the sinusoids. (Original magnification, x25.)

 

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  		Table 1. In Vivo Signal Intensities in Kidney and Liver before and after MSC Injection

 
For ex vivo imaging at 9.4 T, qualitative analysis did not show any signal hypointensity in the control animals whether or not the labeled cells were injected, which demonstrated that neither the nephropathy itself nor cells alone had an effect on the renal signal intensity.

On the contrary, seven of 10 pathologic kidneys showed areas of signal intensity decrease mainly in the cortex (Fig 4), where the glomeruli are located. All cases showed focal areas of signal intensity decrease and involved a maximum of three portions of the kidney. These portions were not necessarily contiguous. In the remaining three animals, no signal intensity decrease was observed after cell injection (Table 2). No differences were noticed on in vivo and ex vivo MR images between the two pathologic groups.



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  		Figure 4a: Ex vivo sagittal three-dimensional T2*-weighted (110/15.7, flip angle of 30°) 9.4-T MR images of (a) control and (b) pathologic kidney 6 days after intravenous injection of 107 labeled MSCs (the upper pole is oriented left). In a, no signal intensity decrease is noted. In b, the corticomedullary differentiation is absent and distinct areas of cortical signal intensity decrease are present in the superior and superior midportion (arrows) poles.

 


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  		Figure 4b: Ex vivo sagittal three-dimensional T2*-weighted (110/15.7, flip angle of 30°) 9.4-T MR images of (a) control and (b) pathologic kidney 6 days after intravenous injection of 107 labeled MSCs (the upper pole is oriented left). In a, no signal intensity decrease is noted. In b, the corticomedullary differentiation is absent and distinct areas of cortical signal intensity decrease are present in the superior and superior midportion (arrows) poles.

 

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  		Table 2. Ex Vivo MR Imaging and Histologic Data

 
Histologic Analysis
Histologic analysis of liver portions showed the presence of numerous Prussian blue stain–positive cells located in the sinusoids (Fig 3), which indicated that many MSCs were trapped by the liver. Among the pathologic kidneys, three showed low glomerular proliferation at hematoxylin-eosin-saffron and {alpha}-actin staining, which indicated a negative or very limited response to the model; in these cases, the absence of Prussian blue stain– and DiI-positive cells indicated an associated low level of MSCs homing to the glomeruli. These animals corresponded to the three negative cases on ex vivo MR images. Two of the animals belonged to the pathologic group in which labeled MSCs were injected 4 days after the induction of the nephropathy and one belonged to the pathologic group in which MSCs were injected 8 days after the induction of the nephropathy.

In the remaining seven pathologic kidneys, analysis of hematoxylin-eosin-saffron staining showed strong diffuse abnormalities in all kidney portions; positive {alpha}-actin staining, Prussian blue staining, and DiI labeling were limited to restricted areas of the tissue (with positive and negative kidney portions). The distribution of the positive histologic areas differed from one kidney to another but involved a maximum of three portions that were not necessarily contiguous. In all kidney portions rated grade 2 or higher for {alpha}-actin, the glomeruli positive for DiI (n = 20) and Prussian blue stain (n = 20) ranged from four to seven and six to eight, respectively (Fig 5). In contrast, in all kidney portions rated less than grade 2 for {alpha}-actin, the glomeruli positive for DiI fluorescence (n = 20) and Prussian blue stain (n = 20) ranged from zero to three and zero to two, respectively (Table 2).



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  		Figure 5a: (a) Micrograph of a pathologic kidney portion shows grade 2 positive area for {alpha}-actin staining, with brown spots corresponding to activated mesangial cells (arrows). (Original magnification, x25.) (b, c) These inflamed areas show a substantial number of Prussian blue– and DiI-positive glomeruli (arrows). Prussian blue– and DiI-positive spots correspond to labeled MSCs. (Original magnification in b, x40; original magnification in c, x25). (d) Analysis in the green channel confirms that bright spots in c are not caused by autofluorescence. (Original magnification, x25).

 


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  		Figure 5b: (a) Micrograph of a pathologic kidney portion shows grade 2 positive area for {alpha}-actin staining, with brown spots corresponding to activated mesangial cells (arrows). (Original magnification, x25.) (b, c) These inflamed areas show a substantial number of Prussian blue– and DiI-positive glomeruli (arrows). Prussian blue– and DiI-positive spots correspond to labeled MSCs. (Original magnification in b, x40; original magnification in c, x25). (d) Analysis in the green channel confirms that bright spots in c are not caused by autofluorescence. (Original magnification, x25).

 


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  		Figure 5c: (a) Micrograph of a pathologic kidney portion shows grade 2 positive area for {alpha}-actin staining, with brown spots corresponding to activated mesangial cells (arrows). (Original magnification, x25.) (b, c) These inflamed areas show a substantial number of Prussian blue– and DiI-positive glomeruli (arrows). Prussian blue– and DiI-positive spots correspond to labeled MSCs. (Original magnification in b, x40; original magnification in c, x25). (d) Analysis in the green channel confirms that bright spots in c are not caused by autofluorescence. (Original magnification, x25).

 


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  		Figure 5d: (a) Micrograph of a pathologic kidney portion shows grade 2 positive area for {alpha}-actin staining, with brown spots corresponding to activated mesangial cells (arrows). (Original magnification, x25.) (b, c) These inflamed areas show a substantial number of Prussian blue– and DiI-positive glomeruli (arrows). Prussian blue– and DiI-positive spots correspond to labeled MSCs. (Original magnification in b, x40; original magnification in c, x25). (d) Analysis in the green channel confirms that bright spots in c are not caused by autofluorescence. (Original magnification, x25).

 
Histologic and MR Imaging Data
Prussian blue stain– and DiI-positive glomeruli were present in the same kidney portions (P < .001) and were located exclusively in kidney portions with a high {alpha}-actin score (P < .001) (Fig 6). Most important, there was a strong correlation between the areas of signal intensity decrease on ex vivo images and the location of {alpha}-actin and Prussian blue stain– and DiI-positive glomeruli (P < .01) (Fig 6).



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  		Figure 6: Graphs show data of hematoxylin-eosin-saffron (HES) staining, {alpha}-actin grading, Prussian blue staining, and DiI-positive glomeruli counting according to the presence (YES) or absence (NO) of signal intensity decrease on 9.4-T MR images in four renal portions: superior (Sup Portion), superior midportion (Mid Sup Portion), inferior midportion (Mid Inf Portion), and inferior (Inf Portion). Results are presented as median (symbols inside boxes) with first and third quartiles (box). Whiskers represent minimum and maximum. Arbitrary units are plotted along the vertical axes. * = P < .01 with Mann-Whitney U test. # = P < .05. High-grade {alpha}-actin areas colocalized with Prussian blue stain–positive areas and DiI-positive glomeruli and corresponded to areas of signal intensity loss on MR images, while no changes were observed for hematoxylin-eosin-saffron staining. This indicates that labeled MSCs specifically target glomeruli with altered mesangial cells and are responsible for signal intensity decrease on MR images.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The results of our study show that MSCs labeled in vitro with a commercially available and clinically approved SPIO formulation (eg, Feridex) and a transfection agent can be successfully injected intravenously and will purposely home to specific injured components (glomeruli) of a target organ (kidney) in previously nonirradiated animals. Moreover, we have demonstrated that these cells can be tracked and precisely localized ex vivo by using a 9.4-T MR system. There was a highly significant correlation between the areas of signal intensity decrease on the ex vivo images and the presence of Prussian blue stain– and DiI-positive cells in these areas. Conversely, in nonpathologic control animals in which labeled cells were injected, no variation in signal intensity was observed on the MR images and no cells were found in the kidney at histologic examination.

Authors of most reports about MR imaging of magnetically labeled stem cells have used local implantation or local grafting of cells into the porcine (19,20) and canine (21) heart and the rat brain and spinal cord (11,12,14–17). These invasive approaches are suitable for only very focal and easily identified lesions but cannot be used in certain organs such as the kidney, in which potentially damaged components (eg, glomeruli or tubules) have a widespread distribution throughout the entire organ. Moreover, proliferative nephropathies are typically focal, and the damaged areas cannot be macroscopically identified. Last, unlike the brain and the spinal cord, stereotactic methods are much more difficult to apply to the kidney because of its intraabdominal location. Our current work differs from previous studies in both the targeted organ and the route of administration of labeled cells. In our study, we chose to inject the cells intravenously for two main reasons: first, because it is the most physiologically relevant technique (ie, endogenous bone marrow stem cells home through the vascular system in the same way) and therefore the most suitable to give us information on how the cells lodge in the glomeruli of the recipient kidney; and second, because it is noninvasive in sick animals with end-stage renal disease.

There are two main mechanisms for homing of stem cells after systemic injection. The first is mainly related to the size of the cells and consists of a passive trapping in the vascular structures of an organ. This has been clearly shown in a previous study where most of the cells directly injected into the renal artery were trapped in the glomerular capillaries of the cortex (22). The second consists of an active recruitment to specific diseased areas. The presence of a decrease in hepatic signal intensity and of numerous Prussian blue stain–positive cells throughout the entire portions of the liver in diseased and control animals in our study calls for an important trapping effect of stem cells into this organ. It remains unclear if this trapping is related to a passive effect, with the sinusoids of the liver acting as a filter, or to a local trophic effect.

Our histologic data clearly show that in pathologic animals, the labeled cells did not target all the glomeruli nonspecifically but homed specifically to the glomeruli that appeared proliferative at hematoxylin-eosin-saffron staining and that showed a high positive grade for {alpha}-actin staining (eg, glomeruli with marked activation of mesangial cells). This suggests that labeled cells are actively recruited by inflammatory glomeruli. To our knowledge, this is the first time that active intraglomerular homing of MSCs could be demonstrated without prior lethal whole-body irradiation. Previous experiments (7–10) with regard to the capability of bone marrow–derived cells to differentiate into glomerular mesangial cells were based on bone marrow transplantation after total body irradiation. Our results suggest that in specific pathophysiologic conditions, MSCs can thus be actively and specifically recruited for the purpose of mesangial repair.

However, the number of cells that localized into the glomeruli was limited, with a maximum of eight out of 20 glomeruli positive for Prussian blue stain and DiI at histologic assessment in inflammatory areas. These findings can explain the absence of signal intensity changes on in vivo images. The small number of cells that homed to the kidney in our study is consistent with the data of Ito et al (9), who noticed that in anti–Thy 1 rats in which green fluorescent protein–enhanced bone marrow cells were transplanted, only 12% of glomerular cells of the restored glomeruli were derived from the transplanted bone marrow.

Technical aspects, especially spatial resolution, could also explain the lack of depiction of this small proportion of cells at in vivo MR imaging. For these in vivo experiments, we prioritized imaging time to limit the duration of imaging for the weak (pathologic) animals. With the two-dimensional sequence, a 1-mm section thickness and 215-µm in-plane resolution were used, which apparently was not sufficient to detect a small number of cells in the kidney. For the ex vivo experiments of the same kidneys at very high field strength (9.4 T), the spatial resolution could be increased with no real limit on the imaging time and the number of signals acquired. This allowed us to dramatically increase the spatial resolution (43 x 63 x 68 µm), as compared with that of the 4.7-T in vivo images, in an imaging time of 7 hours per complete three-dimensional data set. In addition, the T2* magnetic susceptibility effects are expected to increase with the square of the magnetic field, which further enhances the detection at 9.4 T. These very-high-spatial-resolution images allowed us to detect labeled cells in the cortex of pathologic kidneys. Of note is that Hoehn et al (15) achieved high-spatial-resolution three-dimensional fast low-angle shot sequences (78 x 49 x 78 µm3) with a 7-T imager within an imaging time of 1.3 hours, which allowed them to perform in vivo experiments and to detect small amounts of cells in the brain of living animals.

It is important to point out that the hypointense patches seen on the 9.4-T MR images could have been caused by the presence of hemorrhagic areas, induced by the nephropathy itself or by poor perfusion of the kidney after sacrifice. This consideration is unlikely for several reasons. First, we did not detect any signal intensity variation in the kidneys of our control animals, which thus excludes the possibility of the presence of hemorrhage in our model. Second, the kidneys were very thoroughly perfused at the time of sacrifice, and any areas of nonperfusion would have been seen on control animals. Third, no red blood cell accumulation was found at histologic examination. Last, and most important, hemorrhage would not explain the matching of areas of signal intensity decrease on MR images with {alpha}-actin and Prussian blue stain– and DiI-positive glomeruli.

As mentioned earlier, the diffuse characteristics of renal diseases make a local intrarenal injection inappropriate. There are two avenues of injecting cells so that they reach all renal segments: intravenous and intraarterial, and each has advantages and drawbacks. As reported in a previous study with normal kidneys (22), the intraarterial route presents the advantage to selectively target the diseased organ and increase the number of localizing cells, which prevents hepatic sinusoidal trapping and makes it possible to detect and monitor the cells with in vivo MR imaging at 1.5 T. By using T2*-weighted sequences with an imaging time of less than 6 minutes and after intraarterial injection of 6 x 105 labeled MSCs, the authors observed a significant signal intensity decrease in the renal cortex in vivo up to 7 days after injection. Drawbacks of the intraarterial route are that it is invasive, which could be problematic in very weak patients, and, since the cells are passively trapped in glomerular tufts, there is the question of their ability to migrate toward other kidney segments or compartments such as proximal or distal tubules or medulla. In comparison, the intravenous route is easy and safe because of its noninvasiveness. No arterial catheterization, anesthesiology, or contrast medium injection is required, which is advantageous in patients with renal insufficiency. Moreover, findings of the present study showed a selective homing effect, with MSCs being specifically lodged in pathologic glomeruli of the kidney, which consequently allowed a therapeutic effect that is selectively focused on the damaged areas. However, this route of injection has the limitation that only a small proportion of cells reaches the kidney because of the predominant cell trapping in the liver. Increasing the total number of injected cells may lead to cell clumping and induction of microembolisms, with the lungs at particular risk. We did not observe this phenomenon in our study and, at present, it is not clear if this would be of substantial effect in humans.

There were a few limitations in this study. First, the number of rats studied was small, which decreases the statistical power of our assessments. This number was, however, sufficiently large to achieve our main objective: to confidently demonstrate that intravenously injected MSCs can engraft specifically in diseased glomeruli of pathologic kidneys. Second, these cells could not be detected in vivo. This negative result may be circumvented by injecting a larger number of cells or, more important, by increasing the imaging resolution. Third, the fate of SPIO-labeled MSCs within the kidney, in particular the state of differentiation into mesangial cells, was not examined and is currently under investigation, since SPIO labeling may inhibit the differentiation of MSCs into certain specific cell types (34,35).

Cell-based therapy is likely to have many potential applications in the kidney, especially to prevent or delay fibrosis related to various proliferative nephropathies associated with end-stage renal insufficiency, dialysis, and transplantation. The fact that magnetically labeled MSCs injected intravenously can actively and specifically target the damaged components of pathologic kidneys, and that such targeted cellular homing can be depicted with MR imaging, is of great therapeutic importance, whether the cells will be used as replacement cells or as delivery vectors for gene therapy. This selective homing effect may stimulate future systemic cell-based therapy protocols since intravenous injection, despite its limitations, is an easy and minimally invasive procedure, with the therapeutic effect being selectively targeted to damaged areas.


   ACKNOWLEDGMENTS
 
The authors thank Hans-Peter Marti, MD, Division of Nephrology, University Hospital, Zurich, Switzerland, for kindly providing us with monoclonal anti–Thy 1.1 antibody.


   FOOTNOTES
 

Abbreviations: DiI = 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate • MSC = mesenchymal stem cell • SPIO = superparamagnetic iron oxide

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, O.H., J.W.M.B.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, O.H., Y.D., C.C., C.T.W.M., N.G., J.W.M.B.; experimental studies, O.H., E.E.F., R.v.H., C.D., R.X., J.W.M.B.; statistical analysis, C.C.; and manuscript editing, O.H., E.E.F., Y.D., C.C., C.T.W.M., N.G., J.W.M.B.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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